Inbred miniature swine provide a unique preclinical model for the study of transplantation immunity. The present proposal represents the second competitive renewal of the grant which has supported our studies of tolerance to renal allografts in this model over the past ten years. The major objective of these studies has been, and continues to be, to determine the mechanism by which specific tolerance is induced and maintained. The specific aims of the present proposal are to: 1) Determine the role of the thymus in the induction and maintenance of tolerance, as well as its role in breaking tolerance to class I mismatched renal allografts; 2) Characterize tolerance to renal allografts induced by tacrolimus; and 3) Characterize the cells responsible for maintaining tolerance and attempt to transfer tolerance by adoptive transfer. Our previous studies and preliminary data have demonstrated that the induction of tolerance to renal allografts across a selective class I mismatch by a short course of cyclosporin is dependent on an intact thymus and involves induction of regulatory cells, most likely from recent thymic emigrants. Precursors of such regulatory cells in long-term tolerant animals were shown to suppress specific CTL generation in vitro, but only after activation by re-exposure to antigen. We will now characterize these thymic emigrants and regulatory cells in terms of phenotype, spectratype and function (aims 1 and 3). We also present preliminary data indicating that tacrolimus is capable of extending tolerance induction to fully MHC mismatched barriers, a finding which could have important clinical implications. We will now attempt to confirm this result and likewise study its mechanism (aim 2). Finally, during the last project period we have established fully histocompatible miniature swine through further inbreeding, making it possible to utilize adoptive transfer techniques to investigate the mechanism of tolerance for the first time in a large animal model (aim 3). It is hoped that an understanding of the mechanisms by which tolerance is induced and maintained for vascularized transplants in this large-animal model will permit development of appropriate clinical protocols for induction of specific tolerance to organ allografts in human beings.